Plasmon-Assisted Super-Resolution Axial Distance Sensitivity in Fluorescence Cell Imaging

There is currently a great need to develop live-cell compatible optical microscopy tools that can provide super-resolution information on biomolecules, in particular for the study of membrane receptors. We present a novel imaging technique, which employs a nanoplasmonic substrate in combination with conventional confocal fluorescence lifetime microscopy, to deliver an axial position sensitivity of order 10 nm in whole cell imaging. The technique exploits the Purcell effect experienced by fluorescent molecules in the vicinity of noble metal nanoparticles, leading to a reduction of the radiative lifetime and a commensurate increase in fluorescence intensity. We employ this technique to map the topography of the cellular membrane, by imaging the fluorescent protein eGFP labeled to the receptor CXCR4, and further investigate receptor-mediated endocytosis in carcinoma cells. These results demonstrate a new approach in biological cell imaging, using bespoke plasmonic nanostructures to provide axial super-resolution sensitivity, while retaining compatibility with conventional fluorescence microscopy techniques

3D Super-Resolution Imaging with Blinking Quantum Dots

Quantum dots are promising candidates for single molecule imaging due to their exceptional photophysical properties, including their intense brightness and resistance to photobleaching. They are also notorious for their blinking. Here we report a novel way to take advantage of quantum dot blinking to develop an imaging technique in three-dimensions with nanometric resolution. We first applied this method to simulated images of quantum dots and then to quantum dots immobilized on microspheres. We achieved imaging resolutions (fwhm) of 8–17 nm in the <i>x</i>–<i>y</i> plane and 58 nm (on coverslip) or 81 nm (deep in solution) in the <i>z</i>-direction, approximately 3–7 times better than what has been achieved previously with quantum dots. This approach was applied to resolve the 3D distribution of epidermal growth factor receptor (EGFR) molecules at, and inside of, the plasma membrane of resting basal breast cancer cells

NMR Metabolomics of MTLn3E Breast Cancer Cells Identifies a Role for CXCR4 in Lipid and Choline Regulation

The alpha chemokine receptor CXCR4 is up-regulated in certain types of breast cancer. Truncation of the C-terminus of this receptor alters cell morphology and increases invasiveness and metastatic potential. Here, to better understand the effects of CXCR4 expression and truncation in breast cancer cells, we have used high resolution magic angle spinning (HR-MAS) NMR studies of rat breast carcinoma MtLn3E cells to characterize the metabolite complement of cells heterologously expressing human CXCR4 or its C-terminal truncation mutant, Δ34-CXCR4. Notable reductions in choline levels were detected when either cells expressing wild-type CXCR4 or Δ34-CXCR4 were compared with cells containing an empty expression vector. Cells expressing CXCR4-Δ34 had reduced lipid content when compared with either the wild-type CXCR4 expressing cells or those containing the empty expression vector. Taken together, our results show that distinct effects on the metabolite complement can be linked to either CXCR4 expression or CXCR4 regulation. The metabolite markers for these two effects identified in the present study can, in turn, be used to further investigate the role of CXCR4 in metastasis

Cumulative mortality from BCa, other cancer and cardiovascular disease by the highest and lowest categories of CRP, AGC and G/L ratio.

<p>Gray’s test for equality of cumulative incidence functions was performed in presence of competing risks.</p

Hazard ratios (HR) and 95% confidence intervals (95% CI) for risk of death from BCa, overall cancer, cardiovascular disease and all causes by clinical cut-offs of CRP and tertiles of absolute granulocyte count and G/L ratio.

<p>All models were adjusted for age, race-ethnicity, cigarette smoking, alcohol consumption, vigorous physical activity, menopausal status, age of menarche, use of oral contraception and aspirin.</p

Hazard ratios (HR) and 95% confidence intervals (95% CI) for risk of death due to BCa, overall cancer, cardiovascular disease and all causes by continuous levels of clinically detectable CRP, absolute granulocyte count and G/L ratio, and trends from categories of markers, stratified by overweight status and median WC.

<p>All models were adjusted for age, race-ethnicity, cigarette smoking, alcohol consumption, vigorous physical activity, menopausal status, age of menarche, use of oral contraception and aspirin.</p

Characteristics of study participants by status at the end of follow-up.

<p>Characteristics of study participants by status at the end of follow-up.</p

The anti-proliferative effect of anti-EGFR tyrosine kinase inhibitor in combination with mitotane on H295R adrenocortical cancer cells

<p>Introduction: Adrenocortical carcinoma (ACC) is a rare disease with a poor prognosis and limited therapeutic options. Mitotane is considered as a first-line therapy but only 30% of the patients showing an objective tumour response.</p> <p>Erlotinib and gefitinib (tyrosine kinase inhibitors – TKI) inhibit the epidermal growth factor receptor (EGFR), which is highly expressed and occasionally mutated in various cancers. EGFR expression was found to be a good discriminator between malignant and benign adrenal tumours. EGFR mutations in exons 18–21 have been found in 3–10% of ACC cases (but not in the H295R ACC cell line) although their functional significance remains unknown.</p> <p>Aim: The aim of this study was to assess whether erlotinib or gefitinib (used alone or in combination with mitotane) inhibit ACC cell proliferation in a pre-clinical setting.</p> <p>Materials and methods: The proliferation rate of the H295R ACC cell line was assessed by Alamar blue assay: optimal time points for determination of cytotoxic effect of the inhibitors were 72 and 96 h of incubation.</p> <p>Results: Mitotane at a concentration of 10 μM decreased the proliferation rate by 23%.</p> <p>Erlotinib inhibited cell proliferation more effectively than gefitinib, causing a cytotoxic effect of 32 and 43%, vs 6 and 12% for gefitinib, after 72 and 96 h of incubation respectively (P<0.001). The combination of mitotane with the EGFR inhibitors showed an additive effect on cell proliferation (41 and 45% for mitotane+erlotinib, and 29 and 32% for mitotane+gefitinib, at 72 and 96 h, respectively).</p> <p>Conclusions: Erlotinib inhibits cell proliferation more potently than gefitinib, and causes higher H295R cells cytotoxicity in combination with mitotane. Combined therapy with agents targeting the EGFR and standard treatments may have the potential to improve the treatment of ACC patients. Given the lack of somatic mutations in this cell line, the potential mechanism(s) of sensitivity to anti-EGFR TKI is currently being investigated.</p

EGFR-MET interaction is modulated by EGFR mutations.

<p>(A) Western blot (WB) of total MET levels in H1975 derived cells. Tubulin levels are shown as loading control. Values beneath blots are relative levels of MET compared to the total levels in the H1975^L858R/T790M cell line from 2 independent experiments +/−SD (B) MET (7q31) (red signal) copy number analysis by FISH in the three H1975 cell lines using the Leica Kreatech C-MET (7q31)/SE7 FISH probe (KBI-10719). The green signal indicates the chromosome 7 centromere control probe. Scale bar 10 mm. Average copy number and ratio between MET and chromosome 7 centromere probe are also indicated (n = 30 cells). (C) Immunofluorescence of total EGFR (Alexa546 –red in the image) and MET (Cyanine 5 –green in the image) in H1975 derived cells. Hoescht dye was used to stain the nuclei of the cells. Merge panels are also shown. Bars, 20 μm. (D) Co-immunoprecipitation (IP) of EGFR in H1975 derived cell lines. The EGFR antibody was used to immunoprecipitate. EGFR and MET levels are shown in both bound and input fractions. The gels shown in the figure were run separately for the bound and input fractions, as indicated by the dotted line, under the same experimental conditions. (E) Fluorescence lifetime imaging was performed on cells plated to sub-confluence on cover-slips and time-resolved analysis in Tri2. Quantification of average FRET efficiency (*** p < 0.0005) is shown, as well as representative pseudocolour lifetime images showing FRET efficiency and corresponding grayscale donor (EGFR-Alexa 546) and acceptor (MET-Cyanine 5) intensity images. Scale bar: 50μm.</p

<i>In vitro</i> validation of the H1975 EGFR mutant cell lines.

<p>(A) Relative mutant allele frequency was compared in cDNA from each cell line by Digital droplet PCR. (B) WB of total EGFR levels in the H1975^L858R/T790M cell line before and after lentivirus infection with a shEGFR and in the H1975 cell lines. hsc70 levels are shown as loading control. Values beneath blots are relative levels of T-EGFR compared to the H1975^L858R/T790M cell line from 2 independent experiments (C) WB of phospho and total EGFR in H1975 derivative cell lines untreated or treated with EGF (100ng/mL) for 15 min, Erlotinib (1μM, 5μM or 10 μM) for 1 hour or both. hsc70 levels were used as loading control. Quantification of the WB is shown above the figure from 3 independent experiments. Error bars is SD (*p<0.05 and **p<0.001).</p